Antisense oligomers offer great potential as pharmaceutical drugs, as evidenced by the number of antisense drugs currently in clinical development, and aided by the fact that a number of potential limitations of antisense oligomers have been successfully addressed over the past several years (Devi, Stein). Novel uncharged oligomer backbones have been developed to improve uptake into cells, and to increase resistance to nuclease degradation (Hudziak, Iversen, Summerton). For some oligomer structures, for example, morpholino based structures, the modified backbone has been found to give enhanced binding affinity to its target nucleic acid (Iversen, Summerton).
More recently, it has been discovered that a variety of arginine-rich peptides can dramatically increase the level of uptake of uncharged oligonucleotides into cells, including mammalian cells (see, for example, co-owned U.S. patent application Ser. No. 60/466,703, filed Apr. 29, 2003, and corresponding U.S. patent application for “Compositions for Enhancing Transport of Molecules into Cells,” filed Apr. 29, 2004, both of which are incorporated herein in its entirety). This discovery has the potential to significantly increase the therapeutic potential of a variety of antisense oligomers, including those intended to block expression of selected proteins, those aimed at blocking certain donor or acceptor splice sites in pre-processed mRNA, and those designed to treat viral infection by blocking expression of viral genes or replication of single-stranded viral genomes.
In some antisense applications, the optimal targeting sequence against which the oligomer antisense is directed may include a run of four of more cytosine bases, in which case the oligomer will contain a corresponding string of four or more complementary guanine bases. As an important example, an optimal target sequence for the c-myc protein is a region containing the AUG start site of the c-myc RNA that includes a run of four cytosine bases. Anti-sense oligomers directed against the start-codon region of c-myc have a number of important therapeutic applications, including the treatment of cancer, polycystic kidney disease (see, for example, co-owned U.S. Pat. No. 6,875,747, which is incorporated herein in its entirety), coronary-vessel restenosis (see, for example, co-owned PCT patent application WO00/44897, published Aug. 3, 2000, which is incorporated herein in its entirety), and cancer therapy (see, for example, co-owned U.S. patent application US-2003-0087861-A1, published May 8, 2003, which is incorporated herein by reference in its entirety.)
Surprisingly, it has now been found that conjugating an arginine-rich peptide to antisense compounds having runs of four or more guanine bases, in an effort to enhance the cellular uptake of the oligomer, severely compromises the antisense activity of the compound, as well as the ability to purify the compound. Although the basis of this problem is not understood, it appears to involve an interaction between the positively charged peptide and the oligomer compound in a way that promotes formation of G-quartets in the oligomer, thus reducing the solubility and/or ability of the compound to bind to its target nucleic acid. It would therefore be useful to enhance the cellular uptake of such antisense oligomer compounds, by conjugating the compound with an arginine-rich peptide, without degrading the antisense activity of the compound with respect to its intracellular target.
In particular, it would be useful to enhance the cellular uptake of the above c-myc antisense compound without loss of antisense activity, for purposes of enhancing the compound's therapeutic activity in the treatment of cancer, polycystic kidney disease or coronary-vessel restenosis.